1、BRITISH STANDARD BS ISO 11285:2004 Milk Determination of lactulose content Enzymatic method ICS 67.100.10 BS ISO 11285:2004 This British Standard was published under the authority of the Standards Policy and Strategy Committee on 17 September 2004 BSI 17 September 2004 ISBN 0 580 44474 0 National fo
2、reword This British Standard reproduces verbatim ISO 11285:2004 and implements it as the UK national standard. The UK participation in its preparation was entrusted to Technical Committee AW/5, Milk and milk products, which has the responsibility to: A list of organizations represented on this commi
3、ttee can be obtained on request to its secretary. Cross-references The British Standards which implement international or European publications referred to in this document may be found in the BSI Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “S
4、earch” facility of the BSI Electronic Catalogue or of British Standards Online. This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. Compliance with a British Standard does not of itself confer immunity from legal
5、 obligations. aid enquirers to understand the text; present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. Sum
6、mary of pages This document comprises a front cover, an inside front cover, the ISO title page, pages ii to iv, pages 1 to 9 and a back cover. The BSI copyright notice displayed in this document indicates when the document was last issued. Amendments issued since publication Amd. No. Date Comments R
7、eference numbers ISO 11285:2004(E) IDF 175:2004(E)INTERNATIONAL NATSDRAD ISO 15821 FDI 715 tide tsriFino 0-400210-9 Milk Determination of lactulose content Enzymatic method Lait Dtermination de la teneur en lactulose Mthode enzymatique Referecne unbmers OSI 58211002:)E(4 DI:571 F002)E(4 OSI DI dnaF
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11、pteres wetpo erimizf deor irpnti.gn Evyre caneeb sah er taken to sneeru ttah teh file is suitlbae fosu re yb ISO memdob rebeis dna IDF antilano ocmmittees. In the unlikely veent that a problem relating to it is f,dnuo lpsaei enfomr tI ehStneC Olar Secrteiraat ta the serddas igleb nevow. ii BSISO1128
12、5:2004 iiiForeword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for
13、 which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on a
14、ll matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are
15、 circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held re
16、sponsible for identifying any or all such patent rights. ISO 11285IDF 175 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF), in collaboration with AOAC International. It is being published jointly by
17、ISO and IDF and separately by AOAC International. BSISO11285:2004 iv Foreword IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National Committee in every member country. Every National Committee has the right to be represented on the IDF Standing Committ
18、ees carrying out the technical work. IDF collaborates with ISO and AOAC International in the development of standard methods of analysis and sampling for milk and milk products. Draft International Standards adopted by the Action Teams and Standing Committees are circulated to the National Committee
19、s for voting. Publication as an International Standard requires approval by at least 50 % of IDF National Committees casting a vote. ISO 11285IDF 175 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF),
20、 in collaboration with AOAC International. It is being published jointly by ISO and IDF and separately by AOAC International. All work was carried out by the Joint ISO/IDF/AOAC Action Team, Characterization of milk and milk products according to heat treatment, of the Standing Committee on Minor com
21、ponents and characterization of physical properties, under the aegis of its project leader, Mrs E. Lechner (DE). This edition of ISO 11285IDF 175 cancels and replaces IDF 175:1995, which has been technically revised. BSISO11285:2004NITERNATNOIAL STANDARD IS:58211 O4002(E) ID:571 F002(4)E1Milk Determ
22、ination of lactulose content Enzymatic method 1 Scope This International Standard specifies an enzymatic method for the determination of the lactulose content of milk. 2 Terms and definitions For the purposes of this document, the following terms and definitions apply. 2.1 lactulose content mass of
23、substances determined by the procedure specified in this International Standard NOTE The lactulose content is expressed as milligrams per kilogram. 3 Principle Fat and protein are precipitated by the addition of zinc sulfate and potassium hexacyanoferrate(II) solution and are then removed by filtrat
24、ion. Lactose and lactulose are hydrolysed to galactose and glucose, or galactose and fructose, respectively, in the presence of the enzyme -D-galactosidase (-gal). The amount of liberated fructose is stoichiometric with the amount of lactulose: -gal lactose + H 2 O galactose + glucose (1) -gal lactu
25、lose + H 2 O galactose + fructose (2) Lactose is present in milk in significantly higher amounts than lactulose. Thus, the glucose present after hydrolysis would interfere with the determination of fructose. Therefore, the glucose is mostly oxidized by glucose oxidase (GOD) to gluconic acid in the p
26、resence of oxygen: GOD glucose + H 2 O + O 2gluconic acid + H 2 O 2 (3)By this means it is possible to determine small amounts of lactulose in the presence of excess lactose. The hydrogen peroxide formed in reaction (3) is destroyed by catalase. This reaction is used to provide the oxygen which is r
27、equired for the oxidation of the glucose: catalase 2 H 2 O 22 H 2 O + O 2 (4)The remaining unoxidized glucose and the fructose produced by the hydrolysis of the lactulose are phosphorylated by means of adenosine triphosphate (ATP) in the presence of hexokinase (HK) to yield glucose-6-phosphate and f
28、ructose-6-phosphate, respectively. BSISO11285:2004 2 HK glucose + ATP glucose-6-phosphate + ADP (5) HK fructose + ATP fructose-6-phosphate + ADP (6) Glucose-6-phosphate is oxidized by means of NADP +in the presence of glucose-6-phosphate dehydrogenase with consequent production of 6-phosphogluconate
29、 and NADPH. After completion of the reaction, the NADPH formed is measured by means of its absorbance at 340 nm. G-6-P-DH glucose-6-phosphate + NADP +6-phosphogluconate + NADPH + H +(7) Fructose-6-phosphate is isomerized to glucose-6-phosphate in the presence of the enzyme phosphoglucose- isomerase
30、(PGI). The glucose-6-phosphate is oxidized as given in reaction (7). The increase in NADPH is measured by means of its absorbance at 340 nm and is proportional to the fructose and lactulose content. PGI fructose-6-phosphate glucose-6-phosphate (8) In order to compensate for any free fructose which m
31、ay originally be present, a blank determination is performed with the addition of -galactosidase omitted. This blank value is taken into account in the final calculation of the lactulose content of the sample. 4 Reagents Use only reagents of recognized analytical grade. The reagents for the fructose
32、 determination (4.15 to 4.18) are commercially available as test combinations. Take due account of manufacturers instructions. 4.1 Water used in the preparation of the enzyme solutions and buffer solutions shall be fresh and of at least double glass-distilled purity. Water used for other purposes sh
33、all be glass-distilled or of at least equivalent purity. 4.2 Zinc sulfate solution, (ZnSO 4 ) = 300 g/l. Dissolve 30,0 g of zinc sulfate heptahydrate (ZnSO 4 7H 2 O) in water in a 100 ml one-mark volumetric flask (5.2). Dilute to the mark with water and mix. 4.3 Potassium hexacyanoferrate(II) soluti
34、on, (K 4 Fe(CN) 6 ) = 150 g/l. Dissolve 15,0 g of potassium hexacyanoferrate(II) trihydrate (K 4 Fe(CN) 6 3H 2 O) in water in a 100 ml one- mark volumetric flask (5.2). Dilute to the mark with water and mix. 4.4 Buffer solution A, pH 7,5. Dissolve 4,80 g of disodium hydrogenphosphate (Na 2 HPO 4 ),
35、0,86 g of sodium dihydrogenphosphate monohydrate (NaH 2 PO 4 H 2 O) and 0,10 g of magnesium sulfate heptahydrate (MgSO 4 7H 2 O) in 80 ml of water (4.1). Adjust the pH to 7,5 0,1 at 20 C, if necessary, with 1 mol/l sodium hydroxide solution (4.10). Dilute with water to 100 ml and mix (sufficient for
36、 approximately 15 analyses). 4.5 Buffer solution B, pH 7,6. Dissolve 14,00 g of triethanolamine hydrochloride N(CH 2 CH 2 OH) 3 HCl and 0,25 g of magnesium sulfate heptahydrate (MgSO 4 7H 2 O) in 80 ml of water (4.1). BSISO11285:2004 3Adjust the pH to 7,6 0,1 at 20 C, if necessary, with 1 mol/l sodi
37、um hydroxide solution (4.10). Dilute with water to 100 ml and mix (sufficient for approximately 60 analyses). 4.6 Buffer solution C. Dilute 40,0 ml of buffer solution B (4.5) to 100 ml with water and mix (sufficient for approximately 50 analyses). 4.7 Sodium hydrogen carbonate (NaHCO 3 ). 4.8 Hydrog
38、en peroxide (H 2 O 2 ), 30 % (mass fraction). 4.9 Octan-1-ol (C 8 H 18 O). 4.10 Sodium hydroxide solutions, c(NaOH) = 0,33 mol/l and 1 mol/l respectively. 4.11 -D-Galactosidase (-gal), from E. coli (-gal EC 3.2.1.23), 5 mg/ml suspension in 3,2 mol/l ammonium sulfate (NH 4 ) 2 SO 4 solution, 30 units
39、/mg (at 25 C, with lactose as substrate); 300 units/mg (at 37 C, with 2-nitrophenyl-D-galactoside (o-nitrophenyl-D-galactopyranoside) as substrate), respectively. 4.12 Glucose oxidase (GOD), from Aspergillus niger (EC 1.1.3.4), degree of purity II, lyophilizate, 200 units/mg (at 25 C) or 230 units/m
40、g (at 37 C, both with glucose as substrate), respectively. 4.13 Oxidation solution. Dissolve 20 mg of glucose oxidase (4.12) in 1 ml of water. Prepare this solution freshly just before use. 4.14 Catalase, from beef liver (EC 1.11.1.6), suspension of 20 mg/ml in water (stabilized), 65 000 units/mg (a
41、t 25 C; with H 2 O 2as substrate). 4.15 Hexokinase/glucose-6-phosphate-dehydrogenase (HK/G6P-DH), mixture of enzymes from yeast (EC 2.7.1.1 and EC 1.1.1.49), suspension in 3,2 mol/l ammonium sulfate (NH 4 2 SO 4 ) solution, HK and G6P-DH: HK: 2 mg/ml; 140 units/mg (at 25 C; with glucose and ATP as s
42、ubstrate); G6P-DH: 1 mg/ml, 140 units/mg (at 25 C; with glucose-6-phosphate as substrate). 4.16 Phosphoglucose isomerase (PGI), from yeast (EC 5.3.1.9). Suspension in 3,2 mol/l ammonium sulfate (NH 4 2 SO 4 ) solution: PGI: 2 mg/ml 350 units/mg (at 25 C; with fructose-6-phosphate as substrate). NOTE
43、 1 The EC numbers in 4.11, 4.12, 4.14 to 4.16 refer to the Enzymatic Classification number given by the Nomenclature Committee of the International Union of Biochemistry (see 4). NOTE 2 The unit in mentioned 4.11, 4.12, 4.14 to 4.16 (often called the International Unit or Standard Unit) is defined a
44、s the amount of enzyme which will catalyse the transformation of 1 mol of substrate per minute under standard conditions. 4.17 ATP solution Dissolve 50 mg of adenosine-5-triphosphate disodium salt (5-ATP-Na 2 ) and 50 mg of sodium hydrogen carbonate (NaHCO 3 )in 1 ml of water. 4.18 NADP solution Dis
45、solve 10 mg of -nicotinamide adenine dinucleotide phosphate disodium salt (-NADP-Na 2 ) in 1 ml of water. BSISO11285:2004 4 5 Apparatus Usual laboratory equipment and, in particular, the following. 5.1 Analytical balance, capable of weighing to the nearest 0,001 g. 5.2 One-mark volumetric flasks, of
46、 capacity 10 ml, 20 ml and 100 ml. 5.3 Conical flask, with ground glass stopper, of capacity 50 ml. 5.4 Funnel, of diameter 50 mm. 5.5 Fluted filter, medium flow rate, of diameter 125 mm and 70 mm. 5.6 Water bath or drying oven, capable of maintaining a temperature of 40 C 2 C. 5.7 Spectrometer, sui
47、table for making measurements at 340 nm. Spectral line filter photometers suitable for making measurements at 365 nm and 334 nm (mercury lamps) may also be used. The molar absorption coefficients of NADPH (see 9.1) are then 3,4 (365 nm) or 6,18 (334 nm) (lmmol 1 cm 1 ). 5.8 Cuvettes, made of glass o
48、r plastic, with optical path length 10,0 mm. In the case of using plastic cuvettes, check the path length. 5.9 Pipettes, of capacity 20 l, 50 l, 100 l, 500 l, 1 ml, 2 ml, 5 ml and 10 ml, suitable for enzymatic analysis. 5.10 Graduated pipettes, of capacity 2 ml and 10 ml. 5.11 Stirrer rod, for blend
49、ing the cuvette solutions. 6 Sampling It is important that the laboratory receive a sample which is truly representative and has not damaged or changed during transport or storage. Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 707. 7 Preparation of test sample Thoroughly mix the test sample to obtain a representat
